Let-7f靶向抑制A20表达在血管内皮细胞炎症反应中的作用研究  

Role of targeted inhibition of A20 expression by let-7f in the inflammatory response of vascular endothelial cells

作　　者：侯江厚 姚颖杰[1] 詹晓燕[1] 杨奕梅 Hou Jianghou;Yao Yingjie;Zhan Xiaoyan;Yang Yimei(Department of Obstetrics,Kunming City Maternal and Child Health Hospital,Kunming 650031,China)

机构地区：[1]昆明市妇幼保健院产科,650031

出　　处：《中国心血管杂志》2020年第4期351-356,共6页Chinese Journal of Cardiovascular Medicine

摘　　要：目的观察let-7f在氧化低密度脂蛋白(ox-LDL)诱导血管内皮细胞炎症反应过程中的变化规律,探讨let-7f参与动脉粥样硬化起始发病过程的分子机制。方法采用双抗体夹心ELISA方法观察不同剂量ox-LDL处理血管内皮细胞时细胞间粘附分子1(ICAM-1)的表达变化情况,RT-qPCR检测不同剂量ox-LDL处理血管内皮细胞时let-7f的表达变化规律,Western blot法分析不同剂量ox-LDL处理血管内皮细胞时核因子κB(NF-κB)的核移位情况和ox-LDL对人脐静脉内皮细胞A20表达及NF-κB通路活化的影响,双荧光素酶报告实验检测let-7f与A20基因的3’-UTR靶向结合,Western blot法检测let-7f对A20表达的调控。结果 20μg/ml ox-LDL可导致血管内皮细胞培养液中ICAM-1的含量明显升高,血管内皮细胞let-7f的表达明显上调(均为P<0.05),且随着ox-LDL作用剂量的增加,ICAM-1与let-7f的表达变化均呈现明显的剂量效应关系。此外,20μg/ml ox-LDL可导致血管内皮细胞NF-κB向核内移位,随着ox-LDL作用剂量的增加核内NF-κB明显增多(P<0.05)。100μg/ml ox-LDL作用于血管内皮细胞,可引起A20下调,IKK被磷酸化,并进一步导致NF-κB磷酸化增多,而let-7f能够与A20基因的3’-UTR靶向结合并明显抑制其表达。结论 Let-7f可能通过调控NF-κB信号传导通路的活化参与血管内皮细胞的炎症反应过程,提示let-7f可能成为动脉粥样硬化早期预防和治疗的新靶点。Objective To observe the change of let-7f in endothelial cells inflammation induced by the oxidized low-density lipoprotein(ox-LDL)and to discuss the mechanism of let-7f involved in the initial pathogenesis of atherosclerosis.Methods Inflammation was induced by ox-LDL in vascular endothelial cells.Double antibody sandwich ELISA method was used to observe the expression of intercellular adhesion molecule 1(ICAM-1).RT-qPCR was applied to assess the expression of let-7f.Western blot was used to analyze the nuclear translocation of nuclear factor kappa-B(NF-κB)in vascular endothelial cells treated with different doses of ox-LDL.Western blot was used to detect the effect of ox-LDL on A20 expression and activation of NF-κB pathway in vascular endothelial cells.Double luciferase report was used to detect the targeted binding of let-7f with A20 gene 3’-UTR,and Western blot was used to detect the regulation of let-7f on A20 expression.Results Compared with the control group,the concentration of ICAM-1 in the medium of endothelial cells treated with 20μg/ml ox-LDL increased significantly,and the expression of let-7f was increased significantly(both P<0.05).The concentration of ICAM-1 and the expression of let-7f all showed the dose dependent relationship with ox-LDL.20μg/ml ox-LDL stimulated activation of NF-κB pathway in endothelial cells,and NF-κB significantly increased translocation into nucleus in a dose dependent manner(P<0.05).100μg/ml ox-LDL could induce A20 down-regulation,IKK phosphorylation,and further increase NF-κB phosphorylation.Let-7f could target combine with 3’-UTR of A20 gene and significantly inhibit its expression.Conclusions These results suggest that let-7f up-regulation plays an important role in promoting inflammation of endothelial cells through NF-κB pathway activation,and may provide the basis for early prevention and treatment of atherosclerosis.

关 键 词：动脉粥样硬化 氧化低密度脂蛋白 人脐静脉内皮细胞 Let-7f 胞间粘附分子1 

分 类 号：R543.5[医药卫生—心血管疾病]